Gel electrophoresis is used extensively in the field of biotechnology to separate target biological macromolecules (biomolecules), such as DNA, RNA, or protein from a mixture of biomolecules. This analytical tool, which utilizes a gel matrix to achieve separation of biomolecules, is used at the research level and also during the manufacturing and quality assurance testing of biomolecules. It is often desirable to remove a target biomolecules from a gel polymer for further analysis. This invention relates to a device for transferring target biomolecules from the gel matrix to a container, such as an Eppendorf tube, for further analysis.
The excision of gel slices from an electrophoresis gel matrix can be effected by using a razor blade or scalpel to cut away the gel portion corresponding to the target biomolecules and using tweezers or the equivalent to transfer a resultant gel slice to a receiving container. One important objective of a biotechnologist is a high level of experimental result reproducibility. Unfortunately the manual blade technique is susceptible to a high degree of technique related variability, which affects the reproducibility of results obtained in subsequent evaluations of the transferred target biomolecule.
Lack of uniformity of gel slices leads to significant variations in DNA purification yields. In a gel electrophoresis, macromolecules migrate to form bands. The migrations distance of each band in an electric field is determined by a band biomolecule size, shape, mass and charge. If a targeted biomolecule migrates to form a tight band, well separated from other biomolecules a blade excision procedure may be useful to extract a clean sample and repeat the process, if necessary. However, when a band is diffuse and not well separated from other biomolecules, the blade excision procedure has little chance of producing a clean, reproducible sample. Resulting variability can be caused by contaminating biomolecules and/or different isomers exhibiting different degrees of biological activity. In addition, differences in the sizes of excised bands can result in significant variability in follow-up activity assays.
Another disadvantage of the blade technique concerns a safety issue generally associated with sharp objects in general whereby users are susceptible to cuts. In addition, when blades are used to excise gel bands, a further potential health complication exists if the electrophoresis step is carried out with radioactively labelled biomolecules. A cut to a hand during the band excision process could result in a transfer of potentially dangerous radioactive material directly to a user's skin. Still another disadvantage of the blade technique is that it requires a separate means for transferring a blade excised gel slice to a suitable container. Tweezers are commonly used for this transfer where gel slice compression and biomolecule damage can occur. A final disadvantage of the blade technique is that blades and tweezers are generally used repeatedly, thereby potentially leading to contamination of subsequent excised gel slices.
The foregoing problems with a manual blade excision technique demonstrate the need for a user friendly devise for cutting and transferring gel slices to 1) easily extract and transfer a clean reproducible marcromolecule sample; 2) maximize the reproducibility of a band excision process; and 3) protect the user from blade related health dangers. Others have recognized the need for such a devise. In U.S. Pat. No. 6,565,728 Kozulic teaches a device used for cutting and recovering a selected gel piece from a larger gel mass. The gel piece is separated from the gel with a hollow member distal end cutting edge and remains in the hollow member distal end due to reduced hollow member internal pressure caused by the manual movement of a piston. An application of piston pressure decreases the hollow member distal end volume thereby increasing the pressure and releasing the gel piece for transfer to a suitable container for further analysis. In this invention, the gel extraction process is dependent on pressure changes in the gel extraction device. The present invention has no pressure change requirements. Here a sharp cutting edge simply cuts a gel slice which remains in a rectangular hollow receptacle until a manually spring controlled plunger head contact the retained gel slice, causing it to exit the device into a suitable container.
In U.S. Pat. No. 6,393,926, Bowersox teaches a method for sampling solid, liquid, and semi-solid bulk materials including gelatinous material. Here the objective is not to isolate a particular biomolecule but to isolate a sample representative of the mixture as a whole. A spring mechanism is used to retract a device's shaft assembly thereby decreasing an internal pressure which allows an external pressure to keep a selected sample in a collection cavity until the shaft assembly rests increasing internal pressure and releasing the sample. An o-ring is needed to insure collection cavity pressure changes. In opposition to the present invention, as in Kozulic, device internal pressure gradations are needed to both hold and eject a gel sample.
Minden (U.S. Pat. No. 6,342,143) teaches a cutting tool for multiple sample retrieval from electrophoretic gels whereby a spring loaded coupling shaft acts as a piston to move a plunger through a cutting tip to remove a cut band via internal pressure gradations. The spring loaded coupling shaft is connected to a computer in order to control movement of the coupling shaft and plunger. As in the Kozulic patent where a distance between the piston and wall of a lumen cavity is critical for maintaining an appropriate internal pressure, the internal pressure in the Minden patent is highly dependent upon a distance between a moving plunger and an axial passage. Pressure gradations are necessary to operate the device. Since it is unnecessary, the present invention is not designed to change internal pressures. In addition, the present invention does not involve a computer. It requires only one handed operation to efficiently extract clean gel samples in a reproducible fashion.
In U.S. Pat. No. 5,587,062 Togawa, et al. discloses a robotic apparatus for excising gel slices which uses an optical detector to detect gel migration patterns. The device's tubular cutting tool is adapted to be pressed against the gel causing a sliced gel slice to be packed inside the tool. The cutting tool can hold up to four excised gel slices. Ejection of the retained gel slices is achieved with compressed air. Togawa, et al, do not discuss the mechanism by which excised gel slices are retained in the cutting tool. Also, it is not clear whether the gel slices actually remain in the tubular cutting tool until the application of compressed air. However, the disclosure does provide that cutting tool is not designed to be an indispensable part of the invention and that a scalpel can be used to cut out the desired gel portions instead of a cutting tool. In the present invention, the cutting edge and the receptacle for holding the excised gel slices are indispensable parts of the device. The cutting edge is designed to ensure a clean excision of gel slices from a wide variety of gel strengths and thicknesses. The receptacle is designed to ensure proper seating of the excised gel slice to maximize frictional retention forces.
The present invention comprises a gel cutting extraction device capable of controlled one handed cutting and simple removing of clean samples from a wide variety of electrophoretic gels.